Modulation of SERCA-2 expression in human airway smooth muscle reflects phenotypic changes associate with asthma

Oluwaseun Ojo, Katharina Mahn, Tak Lee, Jeremy Ward. MRC and Asthma UK Centre in Allergic Mechanisms of Asthma, King’s College London, London, United Kingdom.

Defects in Ca2+ homeostasis have been implicated in the altered function of airway smooth muscle (ASM) in asthma. Cytosolic [Ca2+] plays a critical role in the function of ASM, not just for contraction but also for regulation of growth, migration and secretion of cytokines. The sarcoendoplasmic reticulum Ca2+ ATPase (SERCA) is an important mechanism for regulating cytosolic [Ca2+], by sequestering cytosolic Ca2+ into the sarcoplasmic reticulum. Our recent studies have shown that SERCA-2 expression is reduced in ASM obtained from asthmatic volunteers, with concomitant changes in ASM Ca2+ handling and proliferation (Mahn et al, PNAS, 2009 In press). We examined the effects of siRNA knockdown of SERCA-2 in ASM obtained from healthy volunteers. The study was approved by the local ethical committee and informed consent obtained.

Methods: ASM cells from healthy and asthmatic volunteers were cultured from endobronchial biopsies (n=10). ASM cells were transfected with siRNA against SERCA-2 or scrambled siRNA, and knockdown efficiency was confirmed by Western blot. Changes in cytosolic [Ca2+] were estimated using fluorescence microscopy and Fura PE3, and ASM proliferation using 3[H]-thymidine incorporation.

Results: ASM treated siRNA against SERCA-2 showed reduced expression of both SERCA-2 message and protein (Western blot) by about 30%; there was a similar reduction in expression of phospholamban, the inhibitory regulator of SERCA-2. As expected, the bradykinin-releasable content of sarcoplasmic reticulum Ca2+ stores was reduced by 50% in ASM transfected with SERCA-2 siRNA compared to scrambled siRNA (p<0.05; n=4 subjects), and the rate constant of recovery of [Ca2+] to baseline after release from stores, a measure of the rate of SERCA-mediated Ca2+ uptake, was decreased from 0.021±0.004 sec-1 (τ = 47 sec) to 0.010±0.002 sec-1 (τ = 103 sec; n=4). ASM cells treated with scrambled siRNA exhibited proliferation when stimulated with 10% fetal bovine serum (FBS) of 285 ± 10% of the unstimulated rate, compared to 417 ± 26% in ASM cells treated with SERCA-2 siRNA (p< 0.02), similar to that ASM derived from asthmatic volunteers (450 ± 90%). Steroids are a mainstay of asthma therapy, and although thought to act primarily by reducing inflammation, are also known to affect transcription of many genes in ASM. Our preliminary studies suggest that treatment of ASM with dexamethasone increases expression of SERCA-2, implying that steroids may provide the added benefit of partially restoring SERCA-2 expression in asthma.

Conclusion: Artificially reducing expression of SERCA-2 in healthy human ASM causes phenotypic changes in Ca2+ handling and proliferation that mimic those seen in ASM obtained from patients with moderate asthma. Reduced efficiency of SERCA-2 function might also be expected to cause disruption of the superficial buffer barrier, leading to increased responses to Ca2+ entry, thus potentially underlying hyperresponsiveness.